The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to human G protein-coupled receptors (GPCRs) which have been altered such that altered GPCRs are constitutively activated. Most preferably, the altered human GPCRs are used for the screening of therapeutic compounds.
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2% or 2,000 genes, are estimated to code for GPCRs. Of these, there are approximately 100 GPCRs for which the endogenous ligand that binds to the GPCR has been identified. Because of the significant time-lag that exists between the discovery of an endogenous GPCR and its endogenous ligand, it can be presumed that the remaining 1,900 GPCRs will be identified and characterized long before the endogenous ligands for these receptors are identified. Indeed, the rapidity by which the Human Genome Project is sequencing the 100,000 human genes indicates that the remaining human GPCRs will be fully sequenced within the next few years. Nevertheless, and despite the efforts to sequence the human genome, it is still very unclear as to how scientists will be able to rapidly, effectively and efficiently exploit this information to improve and enhance the human condition. The present invention is geared towards this important objective.
Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as xe2x80x9cknownxe2x80x9d receptors, while receptors for which the endogenous ligand has not been identified are referred to as xe2x80x9corphanxe2x80x9d receptors. This distinction is not merely semantic, particularly in the case of GPCRs. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed. Thus, the orphan GPCRs are to the pharmaceutical industry what gold was to California in the late 19th centuryxe2x80x94an opportunity to drive growth, expansion, enhancement and development. A serious drawback exists, however, with orphan receptors relative to the discovery of novel therapeutics. This is because the traditional approach to the discovery and development of pharmaceuticals has required access to both the receptor and its endogenous ligand. Thus, heretofore, orphan GPCRs have presented the art with a tantalizing and undeveloped resource for the discovery of pharmaceuticals.
Under the traditional approach to the discovery of potential therapeutics, it is generally the case that the receptor is first identified. Before drug discovery efforts can be initiated, elaborate, time consuming and expensive procedures are typically put into place in order to identify, isolate and generate the receptor""s endogenous ligandxe2x80x94this process can require from between 3 and ten years per receptor, at a cost of about $5million (U.S.) per receptor. These time and financial resources must be expended before the traditional approach to drug discovery can commence. This is because traditional drug discovery techniques rely upon so-called xe2x80x9ccompetitive binding assaysxe2x80x9d whereby putative therapeutic agents are xe2x80x9cscreenedxe2x80x9d against the receptor in an effort to discover compounds that either block the endogenous ligand from binding to the receptor (xe2x80x9cantagonistsxe2x80x9d), or enhance or mimic the effects of the ligand binding to the receptor (xe2x80x9cagonistsxe2x80x9d). The overall objective is to identify compounds that prevent cellular activation when the ligand binds to the receptor (the antagonists), or that enhance or increase cellular activity that would otherwise occur if the ligand was properly binding with the receptor (the agonists). Because the endogenous ligands for orphan GPCRs are by definition not identified, the ability to discover novel and unique therapeutics to these receptors using traditional drug discovery techniques is not possible. The present invention, as will be set forth in greater detail below, overcomes these and other severe limitations created by such traditional drug discovery techniques.
GPCRs share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or xe2x80x9cextracellularxe2x80x9d side, of the cell membrane (these are referred to as xe2x80x9cextracellularxe2x80x9d regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or xe2x80x9cintracellularxe2x80x9d side, of the cell membrane (these are referred to as xe2x80x9cintracellularxe2x80x9d regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The xe2x80x9ccarboxyxe2x80x9d (xe2x80x9cCxe2x80x9d) terminus of the receptor lies in the intracellular space within the cell, and the xe2x80x9caminoxe2x80x9d (xe2x80x9cNxe2x80x9d) terminus of the receptor lies in the extracellular space outside of the cell. The general structure of G protein-coupled receptors is depicted in FIG. 1.
Generally, when an endogenous ligand binds with the receptor (often referred to as xe2x80x9cactivationxe2x80x9d of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular xe2x80x9cG-protein.xe2x80x9d Although other G proteins exist, currently, Gq, Gs, Gi, and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as xe2x80x9csignal transductionxe2x80x9d). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein. A principal focus of this invention is directed to the transmembrane-6 (TM6) region and the intracellular-3 (IC3) region of the GPCR.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an xe2x80x9cinactivexe2x80x9d state and an xe2x80x9cactivexe2x80x9d state. As shown schematically in FIG. 2, a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed xe2x80x9cconstitutive receptor activation.xe2x80x9d
As noted above, the use of an orphan receptor for screening purposes has not been possible. This is because the traditional xe2x80x9cdogmaxe2x80x9d regarding screening of compounds mandates that the ligand for the receptor be known. By definition, then, this approach has no applicability with respect to orphan receptors. Thus, by adhering to this dogmatic approach to the discovery of therapeutics, the art, in essence, has taught and has been taught to forsake the use of orphan receptors unless and until the endogenous ligand for the receptor is discovered. Given that there are an estimated 2,000 G protein coupled receptors, the majority of which are orphan receptors, such dogma castigates a creative, unique and distinct approach to the discovery of therapeutics.
Information regarding the nucleic acid and/or amino acid sequences of a variety of GPCRs is summarized below in Table A. Because an important focus of the invention disclosed herein is directed towards orphan GPCRs, many of the below-cited references are related to orphan GPCRs. However, this list is not intended to imply, nor is this list to be construed, legally or otherwise, that the invention disclosed herein is only applicable to orphan GPCRs or the specific GPCRs listed below. Additionally, certain receptors that have been isolated are not the subject of publications per se; for example, reference is made to a G Protein-Coupled Receptor database on the xe2x80x9cworld-wide webxe2x80x9d (neither the named inventors nor the assignee have any affiliation with this site) that lists GPCRs. Other GPCRs are the subject of patent applications owned by the present assignee and these are not listed below (including GPR3, GPR6 and GPR12; see U.S. Provisional No. 60/094879):
As will be set forth and disclosed in greater detail below, utilization of a mutational cassette to modify the endogenous sequence of a human GPCR leads to a constitutively activated version of the human GPCR. These non-endogenous, constitutively activated versions of human GPCRs can be utilized, inter alia, for the screening of candidate compounds to directly identify compounds of, e.g., therapeutic relevance.
Disclosed herein is a non-endogenous, human G protein-coupled receptor comprising (a) as a most preferred amino acid sequence region (C-terminus to N-terminus orientation) and/or (b) as a most preferred nucleic acid sequence region (3xe2x80x2 to 5xe2x80x2 orientation) transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the GPCR:
P1AA15Xxe2x80x83xe2x80x83(a)
wherein:
(1) P1 is an amino acid residue located within the TM6 region of the GPCR, where P1 is selected from the group consisting of (i) the endogenous GPCR""s proline residue, and (ii) a non-endogenous amino acid residue other than proline;
(2) AA15 are 15 amino acids selected from the group consisting of (a) the endogenous GPCR""s amino acids (b) non-endogenous amino acid residues, and (c) a combination of the endogenous GPCR""s amino acids and non-endogenous amino acids, excepting that none of the 15 endogenous amino acid residues that are positioned within the TM6 region of the GPCR is proline; and
(3) X is a non-endogenous amino acid residue located within the IC3 region of said GPCR, preferably selected from the group consisting of lysine, hisitidine and arginine, and most preferably lysine, excepting that when the endogenous amino acid at position X is lysine, then X is an amino acid other than lysine, preferably alanine; and/or
Pcodon(AA-codon)15Xcodonxe2x80x83xe2x80x83(b)
wherein:
(1) Pcodon is a nucleic acid sequence within the TM6 region of the GPCR, where Pcodon encodes an amino acid selected from the group consisting of (i) the endogenous GPCR""s proline residue, and (ii) a non-endogenous amino acid residue other than proline;
(2) (AA-codon)15 are 15 codons encoding 15 amino acids selected from the group consisting of (a) the endogenous GPCR""s amino acids (b) non-endogenous amino acid residues and (c) a combination of the endogenous GPCR""s amino acids and non-endogenous amino acids, excepting that none of the 15 endogenous codons within the TM6 region of the GPCR encodes a proline amino acid residue; and
(3) Xcodon is a nucleic acid encoding region residue located within the IC3 region of said GPCR, where Xcodon encodes a non-endogenous amino acid, preferably selected from the group consisting of lysine, hisitidine and arginine, and most preferably lysine, excepting that when the endogenous encoding region at position Xcodon encodes the amino acid lysine, then Xcodon encodes an amino acid other than lysine, preferably alanine.
The terms endogenous and non-endogenous in reference to these sequence cassettes are relative to the endogenous GPCR. For example, once the endogenous proline residue is located within the TM6 region of a particular GPCR, and the 16th amino acid therefrom is identified for mutation to constitutively activate the receptor, it is also possible to mutate the endogenous proline residue (i.e., once the marker is located and the 16th amino acid to be mutated is identified, one may mutate the marker itself), although it is most preferred that the proline residue not be mutated. Similarly, and while it is most preferred that AA15 be maintained in their endogenous forms, these amino acids may also be mutated. The only amino acid that must be mutated in the non-endogenous version of the human GPCR is X i.e., the endogenous amino acid that is 16 residues from P1 cannot be maintained in its endogenous form and must be mutated, as further disclosed herein. Stated again, while it is preferred that in the non-endogenous version of the human GPCR, P1 and AA15 remain in their endogenous forms (i.e., identical to their wild-type forms), once X is identified and mutated, any and/or all of P1 and AA15 can be mutated. This applies to the nucleic acid sequences as well. In those cases where the endogenous amino acid at position X is lysine, then in the non-endogenous version of such GPCR, X is an amino acid other than lysine, preferably alanine.
Accordingly, and as a hypothetical example, if the endogenous GPCR has the following endogenous amino acid sequence at the above-noted positions:
P-AACCTTGGRRRDDDE-Q (SEQ.I.D.NO:281)
then any of the following exemplary and hypothetical cassettes would fall within the scope of the disclosure (non-endogenous amino acids are set forth in bold):
P-AACCTTGGRRRDDDE-K (SEQ.I.D.NO.:282)
P-AACCTTHIGRRDDDE-K (SEQ.I.D.NO.:283)
P-ADEETTGGRRRDDDE-A (SEQ.I.D.NO.:284)
P-LLKFMSTWZLVAAPQ-K (SEQ.I.D.NO.:285)
A-LLKFMSTWZLVAAPQ-K (SEQ I.D. NO.:286)
It is also possible to add amino acid residues within AA15, but such an approach is not particularly advanced. Indeed, in the most preferred embodiments, the only amino acid that differs in the non-endogenous version of the human GPCR as compared with the endogenous version of that GPCR is the amino acid in position X; mutation of this amino acid itself leads to constitutive activation of the receptor.
Thus, in particularly preferred embodiments, P1 and Pcodon are endogenous proline and an endogenous nucleic acid encoding region encoding proline, respectively; and X and Xcodon are non-endogenous lysine or alanine and a non-endogenous nucleic acid encoding region encoding lysine or alanine, respectively, with lysine being most preferred. Because it is most preferred that the non-endogenous versions of the human GPCRs which incorporate these mutations are incorporated into mammalian cells and utilized for the screening of candidate compounds, the non-endogenous human GPCR incorporating the mutation need not be purified and isolated per se (i.e., these are incorporated within the cellular membrane of a mammalian cell), although such purified and isolated non-endogenous human GPCRs are well within the purview of this disclosure. Gene-targeted and transgenic non-human mammals (preferably rats and mice) incorporating the non-endogenous human GPCRs are also within the purview of this invention; in particular, gene-targeted mammals are most preferred in that these animals will incorporate the non-endogenous versions of the human GPCRs in place of the non-human mammal""s endogenous GPCR-encoding region (techniques for generating such non-human mammals to replace the non-human mammal""s protein encoding region with a human encoding region are well known; see, for example, U.S. Pat. No. 5,777,194.)
It has been discovered that these changes to an endogenous human GPCR render the GPCR constitutively active such that, as will be further disclosed herein, the non-endogenous, constitutively activated version of the human GPCR can be utilized for, inter alia, the direct screening of candidate compounds without the need for the endogenous ligand. Thus, methods for using these materials, and products identified by these methods are also within the purview of the following disclosure.